| Objective Androgen and its receptor(Androgen receptor,AR)are the key factors for initiating and maintaining benign prostatic hyperplasia(BPH),and the mechanism still needs to be clarified.This study explored the effect and mechanism of androgen on prostate cell proliferation through up-regulation HMGB-1 to mediate autophagy.Methods Adult male SD rats were randomly divided into 4 groups(n = 10 in each group): control group(Ctrl),androgen-induced benign prostatic hyperplasia(TP,testosterone propionate)group,TP+ Chloroquine(CQ,autophagy inhibitor)group and CQ group.Prostate tissues were collected for 21 days.The histopathological changes of prostate were observed by HE staining,the localization and expression of AR and autophagy-related proteins were analyzed by immunohistochemical(IHC)and Western Blot methods,the formation of autophagosomes was observed by transmission electron microscope,and the wet weight,volume and index of prostate were analyzed by measurement to explore that androgen promotes prostate hyperplasia by up-regulating cell autophagy.Human prostatic hyperplasia cells(BPH-1)and human prostatic stromal cells(WPMY-1)were cultured.CCK8,Flow cytometry,Western Blot,MDC and acridine orange staining were used to verify the role of autophagy in androgen-induced prostatic cell proliferation.Secondly,in vivo and in vitro,on the basis of androgen intervention,HMGB-1 and its inhibitor glycyrrhizin(Gly)were given intervention,grouped into Ctrl,TP,HMGB-1,TP+HMGB-1,TP+Gly,Gly,to detect changes in the level of autophagy,and to prove the role of HMGB-1 in androgen-mediated autophagy of prostate cells;repeated verification of HMGB-1 silenced in vitro.Subsequently,the PI3K/AKT signaling pathway blocker Wommanycin(WM)was given to intervene to detect changes in AR,HMGB-1 and key proteins in the signaling pathway,and analyze the mechanism of androgen-induced changes in HMGB-1.Then,Wortmannin(WM),an blocker of PI3K/AKT signal pathway,was given to detect the changes of AR,HMGB-1 and key proteins of signal pathway,and to analyze the mechanism of androgen-induced HMGB-1 changes.Results1.Androgen-mediated autophagy to promote prostatic cell proliferationMale SD rats injected testosterone propionate subcutaneously for 21 days(TP group),prostatic glandular epithelial cells stratified high columnar hyperplasia,part of the interstitial fibrous connective tissue hyperplasia,prostate volume,wet weight and index increased significantly(P < 0.01),indicating the success of the model.At the same time,it was observed that the number of autophagosomes in the glandular epithelial cells of the prostate tissue in the TP group increased,and the expression of AR,HMGB-1,Beclin-1,and LC-3bⅡ/Ⅰ protein increased(P<0.01);the autophagy inhibitor CQ was given intervention,In the TP group,prostate tissue hyperplasia was reduced,prostate volume,wet weight,index decreased,number of autophagosomes decreased,and LC-3bⅡ/Ⅰ protein expression decreased(P<0.05),suggesting that changes in autophagy levels may be involved in androgen promotion The process of prostatic tissue hyperplasia in rats.TP intervention for 24 h can significantly increase the proliferation of BPH-1 and WPMY-1 cells,increase the fluorescence intensity of MDC and acridine orange staining,and increase the expression of AR,HMGB-1,Beclin-1,and LC-3bⅡ/Ⅰ(P<0.05);With CQ intervention,the cell proliferation activity of the TP group was weakened,the fluorescence intensity of MDC and acridine orange staining was weakened,and the expression of LC-3bⅡ/Ⅰ protein was reduced(P<0.05),which verified that androgens can promote prostate cell proliferation by mediating autophagy.Both in vivo and in vitro interventions with TP can increase the expression of HMGB-1 protein in prostate cells,suggesting that HMGB-1 may be a key link in the increase of androgen-induced autophagy in prostate cells.2.The mechanism of androgen mediating prostate cell autophagy through HMGB-1Compared with the Ctrl group,the prostate tissue of the HMGB-1 group was significantly hyperplastic,but the degree of hyperplasia was weaker than that of the TP group(P < 0.05).Compared with TP group,the glandular hyperplasia in TP+HMGB-1 group was more obvious,a large number of glands showed papillary fold protruding to the lumen,the wet weight and index of prostate increased,and the number of autophagosomes in glandular epithelial cells and the expression of HMGB-1,Beclin-1 and LC-3bⅡ/Ⅰ protein were higher in TP+HMGB-1 group(P < 0.05).After the intervention of Gly,an inhibitor of HMGB-1,the hyperplasia of prostate tissue,the volume,wet weight and index of prostate,the number of autophagosomes and the expression of HMGB-1 and LC-3bⅡ/Ⅰ protein were decreased in TP group,suggesting that androgen may mediate autophagy of rat prostate cells through HMGB-1.When BPH-1 and WPMY-1 cells were stimulated by HMGB-1 for 24 hours,the fluorescence intensity of MDC and acridine orange staining and the expression of HMGB-1,Beclin-1 and LC-3bⅡ/Ⅰ proteins were increased in TP group.After Gly inhibition or SI-HMGB-1 transfection of silenced HMGB-1,the autophagy level of BPH-1 and WPMY-1 cells decreased while the expression of HMGB-1 decreased,which verified that androgen can up-regulate autophagy of prostate cells through HMGB-1.In BPH-1 and WPMY-1 cells,compared with Ctrl group,the expression levels of p-PI3 K,p-AKT,AR and HMGB-1 in TP group were up-regulated,and the expression levels of p-PI3 K and p-AKT in TP group were down-regulated while AR and HMGB-1 expression were down-regulated after administration of Wortmannin(WM),an antagonist of PI3K/AKT signaling pathway.Conclusion 1.Androgen can promote prostate tissue proliferation by up-regulating prostate cell autophagy;2.HMGB-1 is one of the ways of androgen up-regulating prostate cell autophagy;3.Androgen may increase the expression of AR and HMGB-1 by activating PI3K/AKT signal pathway. |
